The purpose of this project is to investigate the tumor-suppressive function of SLC5A8 in mammary gland and to evaluate its therapeutic potential in the treatment of breast cancer. SLC5A8 is a plasma membrane transporter originally identified as a tumor suppressor in colon, but neither the identity of its transport function nor how it elicits tumor suppression was known. Our recent studies have not only established the identity of the transport function of this protein but also the mechanism by which the transporter promotes tumor cell-specific apoptosis. SLC5A8 is a Na+coupled transporter for monocarboxylates (e.g., propionate, butyrate, lactate, pyruvate). Butyrate is a well known inhibitor of histone deacetylases (HDACs) and is generated in the colon by bacterial fermentation. HDAC inhibitors preferentially kill tumor cells. We have shown recently that pyruvate is also a potent HDAC inhibitor and that pyruvate and butyrate induce tumor cell-specific apoptosis in a SLC5A8-dependent manner. Thus, the ability of SLC5A8 to mediate the concentrative entry of the HDAC inhibitors pyruvate and/or butyrate into cells underlies the tumor-suppressive function of this transporter. SLC5A8 is expressed in normal mammary gland and the gene is silenced in breast cancer by DNA methylation. Exogenous expression of the transporter in mammary tumor cell lines leads to cell death in a pyruvate/butyrate-dependent manner. Methylation of SLC5A8 gene in mammary epithelial cells involves the DNA methyltransferase DNMT1;activation of the oncogene Ras plays a critical role in this process. Treatment of mammary tumor cells with DNA demethylating agents re-activates SLC5A8 expression. This phenomenon is seen in estrogen receptor (ER)-positive and in ER-negative cells. Importantly, re-expression of SLC5A8 leads to cell death both in ER-positive and ER-negative cells in the presence of pyruvate or butyrate. To achieve a better understanding of how breast cancer cells down-regulate SLC5A8 and how re-activation of the transporter expression leads to tumor cell-specific apoptosis and blockade of mammary tumorigenesis, we propose three aims. Aim 1 is to investigate the mechanism of down-regulation of SLC5A8 in breast cancer. Hypothesis: Methylation-associated silencing of SLC5A8 is secondary to the activation of Ras-associated MAPK signaling pathway. Aim 2 is to confirm the ability of SLC5A8 to block mammary tumorigenesis in vivo. Hypothesis: Re-activation of SLC5A8 in breast cancer or over-expression of SLC5A8 in normal mammary gland will effectively block mammary tumor formation. Aim 3 is to evaluate the therapeutic potential of SLC5A8 in ER-negative breast cancer. Hypothesis: Activation of SLC5A8 expression in ER-negative breast cancer will induce pyruvate/butyrate-mediated tumor cell-specific apoptosis. These studies will generate important and clinically/therapeutically relevant information which will aid in the development of novel strategies for the prevention and/or treatment of breast cancer. PUBLIC HEALTH RELEVANCE: The purpose of this project is to investigate the potential of a newly discovered tumor suppressor as a drug target for the treatment of breast cancer. This tumor suppressor is a plasma membrane transporter which mediates the entry of histone deacetylase (HDAC) inhibitors pyruvate and butyrate into cells. HDAC inhibition causes tumor cell-specific apoptosis and cell death. Breast cancer cells shut off the expression of the transporter to avoid this cell death. In the current project, we will test the hypothesis that re-activation of the expression of the transporter gene in breast cancer cells would induce tumor cell-specific apoptosis through transporter-mediated entry of pyruvate or butyrate. This mode of therapy may be effective not only for the estrogen receptor-positive breast cancer but also for the estrogen receptor-negative breast cancer.